KR20150139673A - Method of and Apparatus for Enriching TAG Contents in Biomass with Glycerol - Google Patents

Abstract

The present invention relates to a method for increasing lipid contents in biomass using glycerol, and an apparatus thereof. The method and apparatus select and culture microalgae having total triacylglycerol contents (TAG) of 3.0 g/L or more, and put glycerol to the cultured microalgae from the outside as a composite nutrient to induce the microalgae to grow by photosynthesis and grow by absorbing the composite nutrient. According to the present invention, if the composite nutrient is input, lipid contents of microalgae is increased 1.3 to 5.2 times more, compared with a case that the composite nutrient is not input.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method and apparatus for enhancing the lipid content of biomass using glycerol,

The present invention relates to a method for improving the lipid content of biomass using glycerol, and more particularly, to a method for improving the lipid content of biomass using glycerol, The present invention relates to a method for improving the lipid content of biomass using glycerol and a device therefor.

Today, efforts are being made to replace conventional petroleum products in various aspects in terms of concern about exhaustion of fossil fuels and prevention of environmental pollution.

What is perceived as the most viable fuel to replace conventional gasoline or diesel is biofuels produced through biomass, a renewable renewable resource. Bioethanol and biodiesel, already used in Brazil and other countries, are recognized as alternative fuels.

Biodiesel is an alternative to petroleum-based diesel oil and is based on renewable resources such as vegetable oils and animal fats. Biodiesel is composed of fatty acid methyl esters and contains two oxygen atoms in the ester functional group. Oxygenated fuel such as biodiesel is advantageous in that the oxygen contained in the molecule during combustion contributes to the combustion reaction, facilitating complete combustion and thus reducing the exhaust gas.

Biodiesel is considered a viable alternative to fossil fuels, which is the most important transportation energy resource, because it can replace current engine oil as a renewable fuel and can be transported and sold through existing facilities. However, biodiesel is still mixed with diesel to improve the low lubrication of pure ultra low sulfur fuel. The production and use of biodiesel is growing rapidly, especially in Europe, the US and Asia, but the share of the total fuel market is still insignificant.

Biodiesel can be produced using rapeseed, grains, farms, corn, etc. However, with the food problems on the planet now unresolved, production of food resources into biomass is not only an efficient use of resources, It is impossible to be free.

From this point of view, the production of biomass using microalgae and the conversion to biodiesel are not, above all, the use of human food resources, so there is no problem in terms of ethics, It is emerging as one of the most promising means of alternative energy.

However, in the case of producing biodiesel using microalgae, the most real problem is that the productivity of microalgae is low, and secondly, the low content of lipid components that can be obtained from microalgae.

At present, the productivity using microalgae is only one fifth to one tenth of that of conventional fossil fuels, which is the weakest point in terms of economy. In recent years, in order to improve such disadvantages, research on development of lipid-high-productivity microalgae using biotechnology has been conducted along with exploration of excellent cell lines, but no remarkable research results have been reported yet.

In addition, it is very important to increase the lipid content of microalgae in order for bio-energy using microalgae to be competitive with existing fossil fuels, and no related technology has been found yet. At present, it is considered that one of the reasons for focusing on improving the productivity of microalgae is a major concern at home and abroad.

Conventional domestic prior art shows a method of extracting and recovering lipids from microalgae as described below, and a technique for increasing the lipid content of microalgae has not been proposed yet.

Korean Patent No. 10-1363723 "Method for Extracting and Recovering Lipids from Microalgae" (Feb. Korean Patent No. 10-1134294 "Oil Extraction from Microalgae and Biodiesel Conversion Method" (Apr. 2, 2012); Korean Patent No. 10-1264543 "Method for extracting raw material oil for biodiesel from microalgae and method for producing biodiesel using the same" (Feb.

Disclosure of Invention Technical Problem [8] The present invention has been made to solve all the problems of the prior art described above, and it is an object of the present invention to provide a microalgae which is capable of increasing the content of lipid components in microalgae, The present invention also provides a method for enhancing the lipid content of biomass using glycerol and its proliferation apparatus.

In order to achieve the above object, the present invention provides a method for producing microalgae, comprising: selecting microalgae and culturing the microalgae in a nutrient medium; The microalgae after completion of the above culturing step are put into a biological reaction vessel and glycerol is added as a combined nutrient source at a rate of 2 g / L to 10 g / L while supplying air to the biological reaction vessel from the outside, Growing the microalgae while kneading for 1 to 20 days; Harvesting the raw material of the biomass by discharging the grown microalgae out of the biological reaction vessel; And extracting a lipid component from the harvested biomass raw material. The present invention also provides a method for improving the lipid content of biomass using glycerol.

In order to perform the method for improving the lipid content of the biomass, the biomass propagation apparatus may be used. The biomass propagation apparatus includes a photobioreactor capable of simultaneously performing independent nutrient growth and heterotrophic growth on the microalgae and further enhancing the lipid content of the microalgae .

In the present invention, one or more of the photobioreactors may be installed, and when a plurality of the photobioreactors are installed in one biomass expansion apparatus, they are preferably installed in a parallel manner.

The production of microalgae by the method according to the present invention can further improve the content of lipids among the constituents of the microalgae, so that even when producing the same amount of microalgae, The production efficiency of algae results in far more substantial results than when the method according to the invention is not used.

In addition, when microalgae are produced using the method of the present invention, the production amount of microalgae is higher than that of the conventional method, so that the practical value that can be utilized as biomass is greater.

1 is a schematic conceptual diagram of an apparatus 100 for growing a biomass according to the present invention,
2 is a schematic conceptual diagram of a photobioreactor 120 used in the biomass propagation apparatus 100,
3 is a schematic conceptual diagram of a light guide plate 126 that can be used in the photobioreactor 120. As shown in FIG.
FIG. 4 is a schematic conceptual view showing a modified embodiment of the biomass propagation apparatus 100,
FIG. 5 is a graph showing measured values according to Table 2,
FIG. 6 is a graph showing measured values according to Table 3. FIG.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. It is to be noted that the accompanying drawings are only for explaining the technical idea of the present invention in detail and that the technical idea of the present invention is not limited thereto and that various modifications are possible.

The method for improving the lipid content of biomass using glycerol according to the present invention comprises culturing microalgae obtained by selecting specific microalgae from microalgae and culturing the selected microalgae in a nutrient medium .

In the present invention, the microalgae are cultivated by selectively sorting only specific species. The present invention preferably uses microalgae having a lipid content of at least 3.0 g / L of microalgae after being grown in a nutrient medium. If the lipid content of the microalgae after the growth in the nutrient medium is too low, the lipid content of the microalgae is relatively low even after feeding and growing the composite nutrient, which is not suitable for use as a raw material for the biomass. The microalgae include both microalgae for drinking water, microalgae for seawater and microalgae for amphibians.

The present invention selects Chlorella sp. As a typical biomass of bamboo storage, selects Nannochloris sp. As a representative biomass for seawater, selects Botryococcus braunii as a representative biomass having both bamboo and sea water properties .

When specific microalgae are selected as biomass, the selected microalgae are cultured in BG-11 medium. The culture conditions of the microalgae were maintained at a temperature of 22 ° C ± 2 ° C during the day (8h) overnight (16h), maintaining the pH of the BG-11 culture solution at 7.2 ± 0.3, To < / RTI > 20 days.

The microalgae that have been cultivated are then transferred to the next step.

In the present invention, the microalgae after completion of the culturing step are introduced into a biological reaction vessel, and glycerol is fed as a nutrient at a rate of 2 g / L to 10 g / L while supplying air to the biological reaction vessel from the outside, And a microbial growth step of growing the microalgae while kneading at room temperature for 12 to 20 days.

The combined micro-algae growth step is a step of introducing the microalgae, which have been cultivated in the previous step, into a biological reaction vessel and inducing the growth of microalgae by photobioreaction. The photobioreaction refers to a process in which microalgae absorb light to perform a photosynthetic action using carbon dioxide present in an aqueous solution, thereby synthesizing proteins and lipids, which constitute the cell tissue of the microalgae. In order to enable the microalgae to perform a uniform photosynthetic action, the photobioreaction generates light by an LED light source from the outside, and in order to uniformly irradiate the microalgae with the generated light, It is preferable to use a cut-out light guide plate.

In the combined micro-algae growth step, micro-algae that have been cultured in the previous step are supplied with carbon dioxide in the air together with the culture solution, and at the same time, glycerol is added as a nutrient at a ratio of 2 g / L to 10 g / L. The glycerol may be added to an aqueous solution containing the culture medium in which the microalgae are grown or the culture thereof in order to provide the microalgae with nutrients necessary for lipid synthesis in addition to the synthesis of cell materials by photosynthesis. When the glycerol is fed at a rate of not less than 2 g / L, the lipid content of the final microalgae is not preferable. Therefore, when the glycerol is fed at a rate of 10 g / L or more, the increase in the lipid content with respect to the input amount is slowed Which is also undesirable.

The micro-algae are uniformly kneaded at room temperature for 12 to 20 days while satisfying the above-mentioned conditions to grow the micro-algae. When the microalgae do not contain a sufficient amount of lipid in the microalgae when they grow to 12 days or less under the above conditions, when the microalgae grow over 20 days or more under the above conditions, Rather than increasing the lipid content proportionally.

The present invention includes a step of performing a combined growth step of the microalgae, and then discharging the microalgae from the biological reaction vessel to harvest the biale as a raw material of the biomass.

The biological reaction vessel is a vessel capable of growing microalgae and discharging the microalgae to the outside, which can be used intermittently in a batch manner or continuously. The method of discharging the microalgae from the biological adaptation vessel to the outside can be carried out by a conventional method.

The present invention includes a step of extracting a lipid component from the biomass raw material of the microalgae harvested in the biological reaction vessel.

The method of extracting the lipid component from the biomass raw material can be carried out by a conventional method. For example, in order to extract the lipids inside the cell wall of the microalgae, the microalgae are dehydrated, dried and the cell walls are disrupted, and then extracted with an organic solvent such as hexane to recover the lipid and residual biomass. At this time, as a method of crushing the cell wall of the microalgae, a high-pressure treatment method, a bead-beating method, a microwave treatment method, an ultrasonic treatment method, an osmotic shock method and the like can be cited. In addition, recently known microalgae can be treated by irradiation with a laser or by using an enzyme.

In order to perform the method for improving the lipid content of the biomass, the biomass propagation apparatus 100 may be used. The biomass propagation apparatus (100) comprises a nutrient supply tank (110) for supporting the microalgae to simultaneously perform independent nutrient growth and heterotrophic growth, And a photobioreactor 120 that can absorb and grow and further enhance the lipid content.

In the biomass propagation apparatus 100, one or two or more photobioreactors 120 may be installed. When a plurality of the photobioreactors 120 are installed in one biomass propagation apparatus, It is preferable to install them in a parallel manner.

1 is a schematic conceptual diagram of an apparatus 100 for growing a biomass according to the present invention,

2 is a schematic conceptual diagram of a photobioreactor 120 used in the biomass propagation apparatus 100,

3 is a schematic conceptual diagram of a light guide plate 126 that can be used in the photobioreactor 120. As shown in FIG.

The present invention preferably utilizes the biomass propagation apparatus 100 in order to efficiently perform a combined growth step of microalgae in which the microalgae after completion of the cultivation step are introduced into a biological reaction vessel and grown.

The biomass propagation apparatus 100 includes a nutrient supply tank 110 for supplying nutrients to the microalgae cultured in the previous stage from the outside and an air supply tank for supplying carbon dioxide in the air to the microalgae 112). The nutrient supply tank 110 stores the nutrient medium and glycerol supplied as the multiple nutrients to the microalgae. The nutrient medium is preferably a BG-11 medium, but not always limited thereto, and a commonly used medium may be used.

The air supply tank 112 supplies carbon dioxide in the air to the microalgae, and the microalgae receive light and supply the nutrients necessary for photosynthesis. The air supply vessel 112 may be directly coupled to the photobioreactor 120 associated with each microalgae and may be indirectly coupled to the bioreactor 120 by being coupled to the nutrient supply vessel 110 have. When supplying the air through the air supply reservoir 112, the CO ₂ gas in the air being held dissolved therein an aqueous solution of the photobioreactor 120, the microalgae using a CO ₂ gas, is supplied from the outside It uses light energy to make photosynthesis. Also, the air supplied to the photobioreactor 120 rises upward while causing bubbles, and in the course of the process, the material necessary for growing the microalgae is kneaded.

The biomass propagation apparatus 100 includes a photobioreactor 120 that allows the microalgae to grow by photosynthesis and allows the microalgae to grow and absorb the nutrients supplied from the outside.

The photobioreactor 120 includes a body 122 that stores an aqueous solution containing nutrients of microalgae and microalgae and is made of a predetermined container, An inlet 123 for supplying the nutrient material and air supplied to the body 122 to the body 122, an outlet 124 for discharging the microalgae after the biological treatment to the outside or discharging the slurry to the outside, A light source unit 125 disposed above the light source unit 125 and generating light by an external power source and a light guide plate 125 for uniformly emitting light generated by the light source unit 125 to the inside of the body unit 122 126).

In the photobioreactor 120, glycerol is supplied as a complex nutrient for promoting growth of the microalgae and improving lipid content. It is preferable to feed the glycerol at a rate of 2 g / L to 10 g / L. When the amount of the glycerol supplied is less than 2 g / L, the growth rate of the microalgae and the lipid content of the microalgae are decreased. When the amount of the glycerol supplied is more than 10 g / L, There is a side where the speed is increased, but it is not preferable because it does not change much compared to the amount of the input. The glycerol is preferably supplied to the microalgae through the nutrient supply tank 110, and the amount of the glycerol to be supplied is determined.

The photobioreactor 120 preferably includes a circulation pump 40. The circulation pump 40 continuously performs kneading and circulation of nutrients and microalgae present in the photobioreactor 120. In addition, the photobioreactor 120 may measure the level of the aqueous solution containing the nutrient through the level gauge 30, and determine whether the nutrient is injected from the outside. It is also preferable to provide various measurement sensors 55 so that the temperature, pH, oxygen or carbon dioxide of the aqueous solution, the concentration of the complex nutrients, and the like can be measured. The water gauge 30 and the measurement sensor 55 may be connected to the computer system 50 to automatically perform measurement and control.

The nutrient medium and the nutrients are introduced into the photobioreactor 120 and the microalgae are introduced into the photobioreactor 120. The light generated from the light source 125 is uniformly dispersed by the light guide plate 126 While slowly kneading for about 12 to 20 days at room temperature. The method of kneading is not particularly limited. The light source 125 generates light using electric energy, and is necessary for the photosynthesis of the microalgae. The light generated by the light source unit 125 is transmitted to the light guide plate 126 disposed below the light source unit 125 and is dispersed evenly inside the body unit 122 by the light guide plate 126. Through such a process, the microalgae grow inside the photobioreactor 120.

The present invention relates to a method of producing glycerol from a microalgae by supplying glycerol as a multiple nutrient externally, not only by a photosynthesis action, but also by a microalgae, It can be said that the growth as a nutritive organism is applied at the same time.

4 is a schematic diagram showing a modified embodiment of the apparatus 100 for growing the biomass.

In the present invention, when the microalgae are cultured in a single species to obtain biomass, it is preferable that the photobioreactor 120 is installed alone.

However, when it is intended to obtain a plurality of biomass by culturing the microalgae into a plurality of species, or if the microalgae are cultured as a single species but want to harvest more biomass within the same period, It is preferable that a plurality of photobioreactors 120 are provided.

When a plurality of the photobioreactors 120 are installed in one biomass expansion apparatus, it is preferable to install them in a parallel manner. In this case, the photobioreactors 120 may be designed to have different capacities or may be designed and used with the same capacity.

In FIG. 4 of the present invention, three photobioreactors 120a, 120b, and 120c are installed at the same capacity, and the photoreflector 120b is installed in a parallel manner.

In this case, the operational relationship of the biomass propagation apparatus 100 is the same as that described above, so that detailed description thereof will be omitted.

Hereinafter, the present invention will be described in more detail with reference to specific examples.

<< Selection and Culture of Microalgae >>

The present invention selected the rectangle Chlorella (Chlorella sp.), And a nano-keulroriseu (Nannochloris. Sp.), And a boat Rio Cocos brownie (Botryococcus. Braunii) as microalgae. The chlorella, nanocholor and barium cocos brownie were cultivated in BG-11 medium and cultivated in Korean Marine Microalgae Bank (KMMCC, Kores). Culturing conditions were proliferated for 15 days in a thermostat (22 ° C ± 2 ° C) with a cycle of pH (7.2 ± 0.3), day (8h) and night (16h). The components of the BG-11 medium used were as shown in Table 1 below.

BG-11 Medium [mg / L Deionized Water] Component Stock Solution
[g / L dH ₂ O] Quantity used Concentration in
Final Medium [M]   Fe Citrate solution - 1 mL -   Citric acid 6 - 3.12 x 10 ^-5   Ferric ammonium citrate 6 - ~ 3.12 x 10 ^-5 NaNO ₃ - 1.5 g 1.72 x 10 ^-2 K ₂ HPO ₄ .H ₂ O 40 1 mL 1.75 x 10 ^-4 MgSO ₄ .H ₂ O 75 1 mL 3.04 x 10 ^-4 CaCl ₃ .H ₂ O 36 1 mL 2.45 x 10 ^-4 Na ₂ CO ₃ 20 1 mL 1.89 x 10 ^-4 MgNa ₂ EDTA and H ₂ O 1.0 1 mL 2.79 x 10 ^-4   Trace metals solution see following recipe 1 mL
- H ₃ BO ₃ - 2.860 g 4.63 x 10 ^-5 MnCl ₂ and H ₂ O - 1.810 g 9.15 x 10 ^-6 ZnSO ₄ .H ₂ O - 0.220 g 7.65 x 10 ^-7 CuSO ₄ .H ₂ O 79.0 1 mL 3.16 x 10 ^-7 Na ₂ MoO ₂ and H ₂ O - 0.391 g 1.61 x 10 ^-6 Co (NO ₃ ) ₂ .H ₂ O 49.4 1 mL 1.70 x 10 ^-7

<< Biomass Proliferation Device (100) >>

The Chlorella sp., Nannochloris sp., And Botryococcus braunii were cultured in BG-11 medium, respectively, and then added to the biomass growth apparatus 100 to measure the growth activity of the microalgae.

4, three photobioreactors 120a, 120b and 120c are installed in the biomass propagation apparatus 100, and the micro-algae are installed in the respective photobioreactors 120a and 120b, (120c), and the initial concentration was measured. The initial concentration of Chlorella sp. Was 0.357 ± 0.7 g / L, the initial concentration of Nannochloris sp. Was 0.367 ± 0.6 g / L, and the initial concentration of Botryococcus braunii was 0.342 ± 0.7 g / L.

Each of the three photobioreactors 120a, 120b and 120c was manufactured in the same shape and the capacity of the body part 122 was 15 L [230 mm (width) * 230 mm (length) * 300 mm (height)]. The photobioreactor 120 was maintained at neutral pH (7.2 ± 0.3), and the progress temperature was 22 ° C. ± 2 ° C., and the cycle of day (8 hours) and night (16 hours) was maintained. In addition, the photobioreactor 120 injected 0.5 L of air per minute into the body portion 112 through the air supply tank 112, and the amount of CO ₂ was 0.02 vvm.

A plurality of measurement sensors 55 connected to the computer 50 are coupled to the photobioreactors 120a, 120b and 120c to measure the state of the aqueous solution of the body part 122 and the water level meter 30 And the circulation of the aqueous solution was continuously induced through the circulation pump 40.

Also, a V-cut type light guide plate 126 is inserted into the body 122 of the photobioreactor 120a, 120b and 120c to uniformly transmit light to the entire reactor. The light guide plate 126 made of PMMA (poly-methylmethacrylate) having the best light transmittance was used as a material, and its thickness was 6 mm.

A light source 125 was installed on the light guide plate 126. The light source 125 used an LED lamp and a white color was used. The amount of light used for photosynthesis was ~250 μmol photon / m ² / s. The LED lamps were manufactured in bar form, and the power supplied to the LEDs was supplied by a model FP-60-12 power supply (AD & Lighting, Suwon, Kyonggi-Do, Korea).

In the case where no glycerol was added as a complex nutrient to the microalgae using the biomass growth apparatus 100 and 2 g / L of glycerol was added as the complex nutrient, 5 g of the glycerol / L was added, and when 10 g / L of glycerol was added, the experiment was conducted.

<< Relationship between the amount of glycerol injected and the experimental method of microalgae >>

While operating the biomass growth apparatus 100, and, the interior of the sample collected in the photobioreactor (120a) (120b) (120c ) per day and the Chlorella sp. And Nannochloris sp. And Botryococcus braunii , and the changes of lipid content.

The experiment was conducted using the three kinds of microalgae obtained from the biomass propagation apparatus 100. In order to calculate the results on the input of glycerol, when the compound nutrient glycerol was not added at all, g / L; 5 g / L; 10 g / L; Of the total, respectively.

<< Correlation between the input of glycerol and the oil extraction amount of microalgae >>

After the biomass propagation apparatus 100 was operated for 20 days, the microalgae were respectively collected and centrifuged at 1500 rpm for 15 minutes. The harvested microalgae were subjected to a wet oil extraction method, (Junsei 96%) were used alone.

Wet oil extraction was performed using 3.3 mL samples containing 1 g of microalgae, and the amount of microalgae was 5% (w / w) relative to the solvent. After the microalgae mixed with the nucleic acid was stirred for 3 hours, the solvent layer containing the oil was recovered and evaporated under reduced pressure (Genevac, EZ2 PLUS). The solvent was volatilized and the remaining oil was recovered. For the complete recovery of the oil, the above extraction procedure was repeated three times.

The amount of oil extracted from the microalgae was calculated as follows.

Chlorella sp. And Nannochloris sp. Were 15.91% and 16.24% at 2 g / L glycerol concentration and Botryococcus braunii were 16.41% at 10 g / L glycerol concentration, respectively.

The values obtained through the above experiment are shown in Table 2 below.

Total TAG content in dry mass of investigated algae species Glycerol
concentration
[g / L]   Total TAG content in dry biomass of algae * (%) Chlorella sp. Nannochloris sp. Botryococcus braunii 0   3.71 ± 0.51   3.13 + - 0.42   7.11 + 1.21 2   15.91 + 1.30   16.24 + 1.40   9.33 ± 1.46 5   8.73 ± 1.43   6.74 + 0.76   15.02 + - 1.67 10   8.21 + - 1.52   6.32 ± 0.67   16.41 ± 1.53

* data from 20-day cell growth was used for determination

Biodiesel is produced by converting TAG (triacylglycerols) contained in microalgae through trans-esterification. Therefore, the more TAG content, the greater the production of biodiesel.

When the method of improving the lipid content of biomass using the glycerol according to the present invention is used, the content of TAG in the case of chlorella is improved from about 3.71% to 8.21% to 15.91% It is possible to obtain a yield of about 2.2 times to 4.3 times or more.

In addition, in the case of the nanoclose, the content of TAG is improved from about 3.13% to 6.32% to 16.24%, so that it is possible to obtain a yield of about 2.0 times to 5.2 times or more.

In addition, in the case of the above-mentioned barium cocos, the content of TAG is improved from about 7.11% to 9.33% to 16.41%, so that the yield of 1.3 to 2.3 times or more can be obtained.

These results are believed to have resulted in an unexpectedly surprising increase in lipid content.

A graphical representation of the measured values according to Table 2 is shown in FIG.

<< Total fatty acid content of microalgae oil >>

The total fatty acid content of microalgae oil can be determined by the composition ratio of saturated fatty acid and unsaturated fatty acid. The lower the constituent ratio of saturated fatty acid and the higher the constituent ratio of unsaturated fatty acid, the higher the stability when biodiesel is used in winter have. When the method of the present invention is used, it is determined whether the constituent ratios of the saturated fatty acids and the unsaturated fatty acids are different from each other, and the total fatty acid content of the microalgae oil is determined in order to confirm whether or not the biodiesel is used stably.

To determine the total fatty acid content of the extracted microalgae oil, a modified direct trans-esterification method was used. 10 mg of dry microalgae and extracted oil were added to each test tube, 2 mL of chloroform-methanol (2: 1, v / v) was added, and the mixture was mixed with a vortex mixer for 10 minutes. Then, the internal standard heptadecanoic acid 1 mL of chloroform solution (Sigma-Aldrich, 500 μg / L), 1 mL of methanol, and 0.3 mL of sulfuric acid. After mixing for 10 minutes with a vortex mixer, the reaction was allowed to proceed for 10 minutes at 100 ° C. After cooling for 10 minutes at room temperature, 1 mL of distilled water was added and mixed for 5 minutes and then centrifuged at 1500 rpm for 10 minutes. The lower layer of the layered solution was extracted with a syringe, filtered and analyzed by GC (Gas Chromatography, Agilent 7890A, Santa Clara, Califonia, USA). Quantitative analysis of fatty acids was performed using Mix RM3, Mix RM5, GLC50, and GLC70 (Supelco) as external reference materials.

The content of free fatty acid in the extracted microalgae oil was measured by acid value analysis by titration method.

The acid value and free fatty acid content were calculated as follows.

Acid value = (56.11 x V x c) / m (2)

   Where V = volume (mL) of the used KOH solution,

        c = molar concentration (M) of the KOH solution,

        m = mass (g) of the sample.

Free fatty acid (%) = 0.5 x Acid value ... (3)

The Chlorella sp. And Nannochloris sp. Were measured in the case of no glycerol input and in the case of glycerol input of 2 g / L, and in the case of Botryococcus braunii , the case of no glycerol input and the glycerol input Was 10 g / L.

The total fatty acid content of the microalgae oil obtained as a result of the experiment was as shown in Table 3 below.

Composition of total fatty acid profiles of algae oil (1)   Fatty acids     Composition (%) of total fatty acids Chlorella sp. Nannochloris sp. Botryococcus braunii Glycerol
concentration [g / L]     0     2     0     2     0     10  Saturated 36.91
± 0.51 34.94
± 0.49 14.27
± 0.34 24.23
± 0.38 18.78
± 0.27 23.39
± 0.42  Unsaturated 63.09
± 2.31 65.06
± 2.48 85.74
± 3.58 75.78
± 3.46 81.24
± 2.61 76.61
± 1.56

* data from 20-day cell growth of medium

As a result of the experiment according to the above table, it was found that the composition ratio of the saturated fatty acid and the unsaturated fatty acid in the microalgae oil slightly varied when glycerol was not added and when glycerol was added.

However, it has been found that the variation is not so large, and the composition ratio of the unsaturated fatty acid is still about 65% to 85%, so that it is still not a problem in using it as a raw material for biodiesel.

A graphical representation of the measured values according to Table 3 is shown in FIG.

<< Fatty acid analysis of microalgae oil >>

In order to confirm the objectivity of the data, we analyzed the fatty acids of the microalgae.

Analysis of fatty acids in microalgae was carried out according to EN ISO 5508 (Animal and vegetable fats and oils - Preparation of test sample) and EN ISO 550 after analysis of fatty acid content (mg / g oil) of extracted oils. 250 mg sample was mixed with 5 mL of methyl heptadecanoate-heptane solution (Fluka, 10 mg / g) as an internal standard and analyzed by GC (Gas Chromatography, Agilent 7890A, Santa Clara, Califonia, USA). The column used for detection and isolation of FAME is capillary Alltech AT-FAME (30 m x 0.25 mm x 0.25 m). Nitrogen was used as the carrier gas. The split ratio of the split mode was 45: 1 and the gas flow rate was 2.2 mL / min. The temperature of the oven was increased to 250 ° C, the temperature of the injector was 250 ° C, and the temperature of the detector was 275 ° C.

The results of analysis of the fatty acids of the microalgae oil are shown in Table 4 below.

Composition of total fatty acid profiles of algae oil (2)  Fatty acids Composition (%) of total fatty acids Chlorella sp. Nannochloris sp. Botryococcus braunii Glycerol
concentration [g / L]    0    2    0    2    0    10 Saturated 36.91
± 0.51 34.94
± 0.49 14.27
± 0.34 24.23
± 0.38 18.78
± 0.27 23.39
± 0.42  C14: 0 mystiric 0.31
± 0.02 0.31
± 0.03 ND 0.58
± 0.04 0.23
± 0.02 ND  C16: 0 palmitic 31.09
± 0.84 15.54
± 0.67 8.62
± 0.48 19.04
± 0.75 9.78
± 0.54 15.23
± 0.38  C17: 0 margarine 0.47
± 0.04 1.71
± 0.11 0.93
± 0.07 0.48
± 0.03 1.55
± 0.14 1.45
± 0.08  C18: 0 stearic 4.11
± 0.12 5.76
± 0.07 2.81
± 0.09 2.85
± 0.06 3.99
± 0.12 5.84
± 0.13  C20: 0 arachidic 0.57
± 0.03 4.13
± 0.24 0.73
± 0.02 0.47
± 0.04 1.37
± 0.03 0.77
± 0.02  C22: 0 behenic 0.24
± 0.02 5.27
± 0.24 1.09
± 0.12 0.71
± 0.07 1.81
± 0.07 0.04
± 0.01  C24: 0 lignoceric 0.13
± 0.02 2.22
± 0.08 0.09
± 0.01 0.1
± 0.04 0.05
± 0.03 0.06
± 0.02 Unsaturated 63.09
± 2.31 65.06
± 2.48 85.74
± 3.58 75.78
± 3.46 81.24
± 2.61 76.61
± 1.56  C16: 1 palmitoleic 2.28
± 0.27 2.1
± 0.38 1.07
± 0.07 2.1
± 0.07 1.66
± 0.03 2.49
± 0.04  C18: 1 oleic 43.01
± 1.14 26.2
± 2.06 62.35
± 2.51 43.47
± 1.67 49.43
± 1.21 49.35
± 1.65  C18: 2 linoleic 10.95
± 1.35 16.76
± 1.31 12.72
± 2.15 17.19
± 1.95 13.67
± 2.11 16.73
± 1.23  C18: 3 linolenic 6.03
± 1.11 8.42
± 1.24 5.44
± 0.48 11.17
± 0.87 10.94
± 1.38 7.03
± 1.41  C20: 1 gadoleic 0.51
± 0.09 2.9
± 0.04 2.12
± 0.03 0.82
± 0.12 4.59
± 0.24 0.75
± 0.07  C22: 1 erucic 0.24
± 0.03 5.63
± 0.12 0.62
± 0.07 0.38
± 0.07 0.83
± 0.13 0.2
± 0.05  C24: 1 nervonic 0.02
± 0.01 3.05
± 0.21 1.49
± 0.56 0.65
± 0.04 0.12
± 0.07 0.05
± 0.01

* ND: not detected; ** data from 20-day cell growth of medium

Since the contents of Table 4 and Table 3 are compared with each other, it is considered that the evaluation according to Table 3 has objectivity.

It can be seen from the above that the fact that the method of the present invention can significantly improve the lipid component of the microalgae has been found objectively.

Now, in order to confirm whether the method of the present invention improves the lipid component of the microalgae and also produces positive results in increasing the production amount of the microalgae , the Chlorella sp. , Nannochloris sp. And Botryococcus braunii under the same conditions and confirmed the results.

<< Correlation between the input of glycerol and the production of microalgae >>

As shown in FIG. 4, three photobioreactors 120a, 120b and 120c are mounted on the biomass propagation apparatus 100, and the photobioreactors 120a, 120b and 120c are connected to the photobioreactors 120a, The internal biomass reactors 120a, 120b and 120c are sampled at the same time every day, while the Chlorella sp., Nannochloris sp. And Botryococcus braunii are injected into the bioreactor 100, .

These experiments were performed on the Chlorella sp., When the compound nutrient glycerol was added at a rate of 2 g / L, when the compound nutrient glycerol was added at the rate of 5 g / L, And nutrient glycerol at a rate of 10 g / L.

As a result, in the case of Chlorella sp. And Nannochloris sp., The effect of glycerol concentration on the amount of biomass was not significant until the 5th day of experiment, but after 5 days, the growth of biomass was remarkable according to the concentration of glycerol Respectively. On the other hand, in the case of Botryococcus braunii , the concentration of glycerol did not significantly affect the amount of biomass until the 7th day of experiment, but it showed a large deviation after 10 days.

As a result of the measurement,

Table 5 for the Chlorella sp., Table 6 for the Nannochloris sp., And Table 7 for the Botryococcus braunii , respectively.

Growth of Chlorella sp. at different glycerol concentration. Glycerol
concentration [g / L]      0      2       5       10 Initial
concentration [g / L]    0.357    0.357    0.357    0.357    1 day 0.36 0.361 0.42 0.38    2 day 0.37 0.39 0.49 0.41    3 day 0.44 0.43 0.53 0.46    4 day 0.47 0.45 0.58 0.48    5 day 0.50 0.50 0.61 0.51    6 day 0.52 0.62 0.78 0.62    7 day 0.55 0.71 0.84 0.75    8 day 0.58 0.82 0.97 0.84    9 day 0.61 0.93 1.14 1.00   10 day 0.64 1.02 1.32 1.24   11 day 0.75 1.24 1.45 1.32   12 day 0.82 1.31 1.51 1.45   13 day 0.91 1.42 1.57 1.53   14 day 1.10 1.45 1.67 1.55   15 day 1.20 1.49 1.75 1.60   16 day 1.24 1.51 1.82 1.63   17 day 1.31 1.53 1.85 1.65   18 day 1.33 1.55 1.88 1.67   19 day 1.35 1.57 1.90 1.70   20 day 1.37 1.61 1.91 1.72

Growth of Nannochloris sp. at various glycerol concentrations. Glycerol
concentration [g / L]      0      2      5      10 Initial
concentration [g / L] 0.367 0.367 0.367 0.367    1 day 0.39 0.52 0.58 0.41    2 day 0.42 0.62 0.68 0.45    3 day 0.45 0.75 0.87 0.48    4 day 0.50 0.91 0.99 0.5    5 day 0.56 1.04 1.23 0.75    6 day 0.61 1.12 1.31 0.92    7 day 0.65 1.19 1.37 1.25    8 day 0.69 1.3 1.41 1.32    9 day 0.75 1.36 1.45 1.40   10 day 0.82 1.42 1.53 1.47   11 day 0.92 1.45 1.61 1.51   12 day 0.97 1.48 1.66 1.56   13 day 1.03 1.51 1.69 1.58   14 day 1.08 1.53 1.71 1.60   15 day 1.10 1.55 1.77 1.62   16 day 1.12 1.57 1.81 1.63   17 day 1.14 1.59 1.86 1.66   18 day 1.16 1.61 1.88 1.68   19 day 1.18 1.64 1.90 1.7   20 day 1.20 1.65 1.92 1.72

Growth of Botryococcus braunnii at different glycerol concentration. Glycerol
concentration [g / L]      0      2      5     10 Initial
concentration [g / L] 0.342 0.342 0.342 0.342    1 day 0.4 0.45 0.42 0.38    2 day 0.49 0.51 0.52 0.48    3 day 0.52 0.60 0.57 0.55    4 day 0.58 0.67 0.64 0.60    5 day 0.62 0.77 0.74 0.67    6 day 0.67 0.84 0.81 0.72    7 day 0.75 0.94 0.91 0.81    8 day 0.80 1.17 1.14 0.90    9 day 0.91 1.52 1.45 1.34   10 day 1.00 1.72 1.69 1.50   11 day 1.06 1.77 1.71 1.61   12 day 1.10 1.80 1.76 1.68   13 day 1.15 1.85 1.81 1.72   14 day 1.20 1.91 1.86 1.76   15 day 1.25 1.97 1.90 1.82   16 day 1.30 2.14 2.06 1.84   17 day 1.34 2.25 2.12 1.85   18 day 1.45 2.29 2.21 1.85   19 day 1.46 2.30 2.25 1.86   20 day 1.47 2.31 2.24 1.87

As a result of analyzing the above data, it was found that more microalgae could be harvested when glycerol was added, compared to the case where glycerol was not added to each microalgae.

In addition, according to the method of the present invention, the present invention significantly improves the content of lipid components in microalgae and at the same time brings about a considerable increase in the production of microalgae .

While the present invention has been particularly shown and described with respect to the preferred embodiments thereof, it is to be understood that the invention is not limited thereto, And the range is determined and limited by the claims.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope of the present invention.

30: water gauge, 40: circulation pump,
50: computer 55: measuring sensor,
110: nutrient supply tank, 112: air supply tank,
120: photobioreactor, 122: body part,
123: inlet, 124: outlet,
125: light source part, 126: light guide plate,

Claims (6)

Selecting a microalgae and culturing the selected microalgae in a nutrient medium;
The microalgae after completion of the culturing step are put into a biological reaction vessel and glycerol is added as a complex nutrient at a rate of 2 g / L to 10 g / L while supplying air to the biological reaction vessel from the outside, Growing the microalgae while kneading for 12 to 20 days;
Discharging the grown microalgae from the biological reaction vessel to harvest the biomass as a raw material;
Extracting a lipid component from the biomass raw material of the microalgae harvested in the biological reaction vessel; To
Wherein the lipid content of the biomass is increased by using glycerol.
The method according to claim 1,
The growth and harvesting of the microalgae is performed using the biomass propagation apparatus 100,
The biomass propagation apparatus (100) comprises a nutrient supply tank (110) for supporting the microalgae to simultaneously perform independent nutrient growth and heterotrophic growth, And a photobioreactor (120) that allows the lipid content to be further improved after absorption and growth,
In the biomass propagation apparatus 100, one or two or more photobioreactors 120 may be installed. When a plurality of the photobioreactors 120 are installed in one biomass propagation apparatus, A method for improving the lipid content of biomass using glycerol.
3. The method of claim 2,
The photobioreactor 120 includes a body 122 made of a predetermined container for growing microalgae,
An inlet 123 for supplying nutrients and air supplied from the nutrient supply tank 110 and the air supply tank 112,
An outlet 124 for discharging the biologically processed microalgae to the outside or discharging the inside slurry to the outside,
A light source unit 125 generating light by an external power source and positioned above the body unit 122,
And a light guide plate (126) for uniformly emitting light generated by the light source unit (125) to the inside of the body part (122). The method for improving the lipid content of biomass using glycerol.
4. The method according to any one of claims 1 to 3,
Wherein the microalgae is any one or two or more selected from the group consisting of Chlorella sp., Nannochloris sp., And Botryococcus braunii . The lipid content of biomass using glycerol .
In an apparatus for growing and harvesting microalgae,
A nutrient supply tank 110 for storing and supplying nutrients of microalgae;
An air supply tank 112 for supplying carbon dioxide (CO ₂ ) in the air to the microalgae;
A plurality of photobioreactors 120 that utilize nutrients supplied from the nutrient supply vessel 110 and induce photosynthesis by using a self photosynthesis system; And,
The photobioreactor 120 includes a body 122 made of a predetermined container for growing microalgae,
An inlet 123 through which the nutrient supplied from the nutrient supply vessel 110 is introduced into the body 122,
An outlet 124 for discharging the biologically processed microalgae to the outside or discharging the inside slurry to the outside,
A light source unit 125 generating light by an external power source and positioned above the body unit 122,
A light guide plate 126 uniformly emitting the light generated by the light source 125 to the inside of the body 122,
And a circulation pump (40) for continuously kneading and circulating nutrients and microalgae present in the body part (122), thereby increasing the lipid content of the biomass using glycerol.
6. The method of claim 5,
The photobioreactor 120 is provided with two or more,
Wherein the plurality of photobioreactors (120) are installed in a parallel manner. The apparatus for increasing the lipid content of biomass using glycerol.

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